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WO1994005267A2 - Utilisation de carraghenanes comme antagonistes de facteur de croissance - Google Patents

Utilisation de carraghenanes comme antagonistes de facteur de croissance Download PDF

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Publication number
WO1994005267A2
WO1994005267A2 PCT/GB1993/001848 GB9301848W WO9405267A2 WO 1994005267 A2 WO1994005267 A2 WO 1994005267A2 GB 9301848 W GB9301848 W GB 9301848W WO 9405267 A2 WO9405267 A2 WO 9405267A2
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cells
carrageenan
growth factor
bfgf
carrageenans
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WO1994005267A3 (fr
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Richard Michael Hoffman
Dietrich Herbert Walter Paper
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Medical Research Council
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Medical Research Council
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Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients

Definitions

  • This invention relates to growth factor antagonists, compositions comprising said antagonists, and a method of making compositions suitable for treatment of certain diseases.
  • polysaccharides exhibit extreme diversity in their
  • polysaccharides are natural compounds obtainable from seaweeds, such as the carrageenans and laminarins.
  • the carrageenans are a family of sulphated polysaccharides which may be isolated from red algae.
  • the backbone structure of most carrageenans consists substantially of repeating galactose and 3, 6 anhydrogalactose residues linked beta-1,4 and alpha-1,3 respectively.
  • capillaries or "neovascularisation" appears to play a key role in the growth and development of solid tumours.
  • substances which inhibit angiogenesis might be able to inhibit the growth of certain solid tumours.
  • Tanaka et al. have shown that a bacterial
  • SP-PG sulphated polysaccharide peptidoglycan complex
  • WO 88/05301 discloses anti-metastatic properties for a number of sulphated polysaccharides, including a type of carrageenans called carrageenan lambda. Thus it was suggested that carrageenan lambda inhibited the spread (but not the growth) of tumours. The mechanism shown to be responsible for this property was inhibition of
  • FGF Fibroblast Growth Factor
  • TGF-beta Transforming Growth Factor-beta
  • the invention provides a compound being a carrageenan or derivative thereof for use as a growth factor suppressor or inhibitor.
  • cne growth factor suppressed or inhibited is one which binds heparin.
  • carrageenans also inhibit growth factors by means other than or in addition to direct competition for binding, thus the present invention extends beyond use of carrageenans or their derivatives to inhibit growth factors to which they bind.
  • carrageenans have been found by the present inventors not to inhibit binding of TGF-alpha to target cells but nonetheless to inhibit TGF-alpha-mediated DNA synthesis.
  • the growth factor suppressed or inhibited by the carrageenan or its derivative will be basic Fibroblast Growth Factor (bFGF), Platelet-derived Growth Factor
  • PDGF Transforming Growth Factor-Beta
  • TGF-beta Transforming Growth Factor-Beta
  • the carrageenans of the present invention will have very large molecular weights, typically over 100,000 Daltons. However it is probable that lower molecular weight derivatives are obtainable which also possess desirable anti-tumour activities. For example, one might expect certain derivatives of the carrageenans (such as oligosaccharides) to have similar properties. This has been shown for low molecular weight derivatives of heparin (Ishihara et al., 1992, Analytical Biochemistry 202, 310-315).
  • derivatives of carrageenans may be prepared in a number of ways.
  • lower molecular weight derivatives may be prepared by chemical or enzymatic hydrolysis using mild acid or carrageenase (respectively) to produce a range of compounds.
  • Such methods will also produce substances with a range of degree of sulphation.
  • product fractions can be screened for activity. Once the most potent fractions have been identified, these derivatives will probably be preferred for therapeutic use (rather than the intact carrageenan) as they will be more
  • Another method of producing lower molecular weights derivatives is by freeze-drying, described below.
  • carrageenans of the present invention are those obtainable commercially, such as the types known as iota, lambda and kappa.
  • inhibiting activity might possess anti-tumour activity.
  • the present inventors have found this to be the case.
  • the carrageenans of the present invention are thought to exert anti-tumour activity by at least two distinct mechanisms (described below). It is a highly preferred feature that the carrageenan or derivative thereof exhibits anti-tumour activity, preferably anti-tumour growth activity.
  • psoriasis which, as demonstrated by Elder et al., [1989, Science 243, 811-814], is associated with the over-production of TGF-alpha
  • pyogenic granuloma pyogenic granuloma
  • alopecia Epidermal Growth Factor [EGF]
  • EGF Epidermal Growth Factor
  • TGF-alpha TGF-alpha have both been shown to inhibit hair follicle elongation [Philpott et al., 1992, British Journal of Dermatology 127, 600- 607], and inhibition of inappropriate keratinocyte proliferation (which is stimulated by TGF-alpha).
  • composition for use in the treatment of tumours and/or of diseases associated with aberrant production of growth factors and/or diseases involving inappropriate
  • composition comprising a
  • compositions according to the invention will be particularly effective in the treatment of solid tumours such as brain tumours (gliomas), and prostate gland cancers.
  • solid tumours such as brain tumours (gliomas), and prostate gland cancers.
  • angiogenesis-dependent diseases which may be susceptible to treatment with compositions according to the invention are in the areas of: ophthalmology
  • fibroplasia fibroplasia
  • cardiology anterior- and hypothalamic lesions
  • paediatrics paediatrics
  • Other conditions which might be usefully treated include: Osier-Weber Syndrome, Nonunion fractures, arteriovenous malformations, arthritis, scleroderma, vascular adhesions, hypertrophic scars and delayed wound healing.
  • the invention provides a method of treating a human or animal body comprising
  • compositions in accordance with the invention comprising administering an effective amount of a composition in accordance with the invention.
  • the formulation of the composition, and the route of delivery, will depend on the disease or condition to be treated. For example, in treating solid, internal
  • tumours one could inject or infuse a sterile, isotonic, aqueous solution of a carrageenan salt or derivative in a neutral pH buffer (such as phosphate-buffered saline).
  • a neutral pH buffer such as phosphate-buffered saline
  • the composition could be given orally (e.g. for the treatment of cancers of the gastrointestinal tract).
  • the ideal formulation would then be a viscous, aqueous solution or gel containing a carrageenan salt (or derivative).
  • the composition could be administered as eyedrops (in a sterile, isotonic, aqueous solution).
  • the composition could take the form of a
  • carrageenan per kilogram body mass more especially in the range 2-20 mg per Kg body mass.
  • Figure 1 is a graph of bFGF binding against concentration of carrageenan
  • Figure 2 is a graph of receptor bound TGF beta 1 against concentration of carrageenan
  • Figure 3 is a graph of specific binding against concentration of lambda carrageenan or iota carrageenan
  • Figure 3b is a graph of cpm against concentration of iota carrageenan
  • Figure 4 is a graph of PDGF bound against concentration of carrageenan or heparin
  • Figure 5 is a graph of radiolabelled bFGF bound against concentration of unlabelled bFGF
  • Figures 6a-6d are graphs of bFGF bound against
  • Figures 7a and 7b are graphs of bFGF bound against concentration of heparin
  • Figure 8 is a graph of bFGF bound against time (in minutes).
  • Figures 9a-9c are graphs of cell numbers against time (in days);
  • Figures 10a-10f are graphs of cell proliferation against concentration of carrageenan
  • Figure 11 is a graph of cell proliferation (as a
  • Figure 12 is a graph of optical density against time (in days).
  • Figures 13a-13d are graphs of DNA synthesis against concentration of carrageenan
  • Figure 14a is a bar chart showing DNA synthesis in LNCaP cells under various conditions
  • Figure 14b is a graph of relative thymidine incorporation (as a percentage of the control) against concentration (in ug/m) of iota carrageenan;
  • Figure 15 is a picture showing the results of
  • Figure 16a and 16b are graphs of thymidine incorporation against concentration of carrageenan for MCF-7 and T47D cells respectively;
  • Figures 17a and 17b are graphs of O.D. against
  • Figure 18 is a graph of relative thymidine incorporation (%) against concentration of iota carrageenan
  • Figure 19 is a bar chart showing thymidine incorporation under different conditions
  • Figures 20a-d are graphs of relative thymidine
  • Figure 21 is a graph of clotting time against
  • Figures 22 and 23 are bar charts showing bFGF binding and DNA synthesis respectively occurring in FBHE cells in the presence of different iota carrageenan derivatives.
  • BHK cells Baby hamster kidney cells
  • antibiotics 100 units/ml penicillin and 100 ug/ml streptomycin.
  • Figure 1 is a graph showing binding of 0.1nM radiolabelled bFGF (as a percentage of positive controls) against concentration of carrageenan (in ug/ml). Determinations were made after a 3 hour incubation at 4°C. The order of potency of antagonistic effect was iota (most, filled circles on the graph) lambda (intermediate, triangles), kappa (least, boxes), with IC50 values of 0.4, 2.4 and 5.7 ug/ml
  • IGF1 >200 >200 >200
  • TGF beta 1 Another growth factor investigated for inhibition by carrageenans was TGF beta 1. Binding of radiolabelled TGF beta 1 ([ 125 I] TGF beta 1, purchased from NEN [Du Pont UK Limited, Stevenage, Hertfordshire]) to Swiss 3T3 cells was determined by the method of Wakefield (1987, Methods
  • Figure 2 is a graph of receptor-bound TGF beta 1 (as a percentage of positive controls) against concentration of carrageenan (in ug/ml).
  • the carrageenans did inhibit binding of TGF beta 1 to Swiss 3T3 cells, but this inhibition was weaker than the inhibition of bFGF binding to BHK cells.
  • IGFl Insulin-like Growth factor 1
  • L23/P cells a large cell adenocarcinoma line
  • FCS penicillin/streptomycin
  • L23/P cells were plated out at 1 ⁇ 10 cells/well in 24 well plates and grown to 90% confluency (24 hours). Cells were washed once with binding buffer
  • Figure 3 shows that carrageenan lambda did not inhibit binding of IGFl to L23/P cells at concentrations up to 250 ug/ml. Similarly, carrageenans kappa and iota failed to inhibit binding of IGFl at concentrations of 200 ug/ml (data not shown). Thus the carrageenans do not behave in the same way as suramin with regard to all growth factors (suramin inhibits TGF-beta 1).
  • Figure 3 also shows data (filled circles) showing that iota carrageenan does not inhibit binding of lnM[ 124 I] TGF-alpha to A431 cells. Further experiments, using standard thymidine
  • iota carrageenan was able to inhibit DNA synthesis stimulated by bFGF or TGF-alpha, but not IGFl.
  • bFGF binds to both low and high affinity receptors on BHK cells, with the low affinity site probably representing heparin or heparin-like substances (Moscatelli, 1987). Binding of bFGF to low and high affinity sites on BHK cells can be distinguished by first washing the cells with 2M NaCl at pH 7.5 and then by extracting the cells with 0.5% Triton X-100 in 0.1M sodium phosphate, pH 8.1
  • concentrations of unlabelled bFGF reduced binding of radiolabelled bFGF to Triton X-100 extractable sites.
  • Figures 6a-6d compare the effects of carrageenans and heparin on NaCl-extractable and triton-extractable bFGF respectively (Key as for Figure 5).
  • Carrageenan iota (Fig. 6a) and heparin (6d) were about equally potent at antagonising binding to the NaCl-extractable (low
  • FIG. 8 shows the displacement of bFGF by CAR/I (circles) or by heparin (boxes) from tritonextractable (high affinity, closed symbols) and NaCl (low affinity, open symbols) sites respectively.
  • Carrageenan iota and heparin exhibited similar activities causing maximum displacement of bFGF from low affinity sites within the first 15 minutes and slower displacement from high affinity sites.
  • FBHE cells were purchased from ECACC and were maintained in DMEM supplemented with 10% FCS, antibiotics (100 units /ml penicillin and 100 ug/ml streptomycin) and supplied with bFGF (10 ng/ml every alternate day).
  • Figures 9a - 9C are graphs of optical density (O.D., a measure of cell numbers) against time for starting concentrations of 5 ⁇ 10 3 cells/ml, 10 /ml and 2 ⁇ 10 4 /ml respectively.
  • FBHE cells were plated out at these different concentrations and growth in the presence and absence of bFGF was compared. Growth in the presence of bFGF was linear up to 8 days for cells plated out at 5 ⁇ 10 3 /ml and 10 4 /ml ( Figures 9a, 9b). Growth was reduced in the absence of bFGF.
  • An anti-bFGF antibody reduced the growth of the FBHE cells grown in the absence of exogenously added bFGF by
  • Carrageenan iota (CAR/I) was found to be a potent
  • CAR/I or CAR/L Basic FGF-independent growth was also inhibited by the carrageenans.
  • the carrageenans reduced this growth by up to 63%. This is greater than growth reduced by an anti-bFGF antibody, indicating that the carrageenans may inhibit another growth factor which stimulates FBHE cell proliferation.
  • the time course of inhibition of proliferation by CAR/I was examined by plating the cells out at 10 3 /well and determining cell proliferation at two-daily intervals.
  • Figure 11 is a graph of cell proliferation (as a percentage of positive controls) against concentration of CAR/I (in ug/ml) for cells treated with iota carrageenan for 2 hours (filled
  • Figure 12 which is a graph of O.D. against time (in days) shows FBHE cell proliferation in the presence (boxes) or absence (circles) of CAR/I (at 40 ug/ml) in the presence (closed symbols) or absence (open symbols) of 10ng/ml bFGF. Error bars represent the standard deviation of triplicate determinations and are smaller than the symbol not shown. This was confirmed by staining the cells with trypan blue (data not shown).
  • FBHE cells or NRK cells
  • MDMEM MDMEM
  • Figures 13a-d are graphs of DNA synthesis (as a percentage of controls) against concentration of CAR/I (13a), CAR/K (13b) and CAR/L (13c) respectively.
  • CAR/I and CAR/L were potent antagonists of bFGF-stimulated DNA synthesis and CAR/K was a weaker antagonist.
  • Basic-FGF-independent DNA synthesis was also inhibited by the carrageenans.
  • Figure 13d is a graph showing inhibition of DNA synthesis in NRK cells by iota carrageenan.
  • FBHE cells were either left unstimulated (open circles) or were stimulated with bFGF (closed circles) and treated simultaneously with the various carrageenans.
  • NRK cells were either left unstimulated (open circles) or stimulated with 5% serum (closed circles). DNA synthesis was determined 8 hours later with a 16 hour exposure to thymidine. Error bars represent the SD of triplicate determinations and if not shown are smaller than the symbol.
  • the foregoing examples demonstrate that the carrageenans inhibit the binding to target cells of hepar in-binding growth factors: bFGF (Example 1); TGF beta 1 (Example 2); and PDGF (Example 4), but do not inhibit the binding of IGFl (Example 3) or TGF alpha (Hoffman, 1993, cited previously).
  • bFGF is inhibited from binding to both low and high affinity receptors (Example 5) and this inhibition is probably the result of iota carrageenan binding to the heparin-binding domain on bFGF rather than binding to the high affinity receptor (Example 5, and Hoffman & Sykes, 1993, Biochemical Pharmacology 45, 2348-2351).
  • L23/P is a non-small cell lung carcinoma (NSCLC)
  • L23/R is a multi-drug resistant variant of L23/P.
  • BEN and LUDLUl are 2 human squamous NSCLC cell lines.
  • BEAS-2B is a non-tumourigenic bronchial epithelial line.
  • Swiss 3T3 is the well-known mouse fibroblast line.
  • MCF-7 and T47D cells are both breast cancer cell lines.
  • U87MG and G36 are both glioma cell lines.
  • LNCaP lymph node carcinoma of the prostate
  • PC3 is an androgen-independent prostate cell line.
  • A375 is a melanoma cell line.
  • Swiss 3T3 cells were maintained in DMEM/10% NBCS.
  • PC-3 cells were maintained in Ham's F12, 1% AA, 1% NEAA.
  • NRK cells were maintained in DMEM, 5% FCS, 1% NEAA. All media were supplemented with antibiotics (100 units/ml
  • Cell proliferation was assessed by performing an MTT assay (as described previously). Briefly cells were plated in 96-well microtitre plates (2000 cells/well, except L23/P plated at 1000 cells/well, and BEN and LUDLUl, plated at 4000 cells/well) overnight. Fresh medium containing carrageenan was then added and the numbers of viable cells were determined by an MTT assay 5d later according to the published method of Twentyman and Luscombe (1987, Brit. J. Cancer 56, 279-285).
  • DNA synthesis was determined by means of a thymidine incorporation assay. Essentially cells were plated in 96-well microtitre plates (4000 cells/well, except L23/P plated at 2000 cells/well, and BEN and LUDLUl plated at 8000 cells/well) for 1-2d depending on the cell line.
  • Table 3 illustrates that DNA synthesis in LNCaP cells is particularly susceptible to inhibition by iota
  • bFGF growth factor bFGF
  • bFGF is one of a number of growth factors implicated in the growth regulation of prostate cancer lines (reviewed by McKeehan, 1991, Cancer Surveys 11, 165-175).
  • the effect of excess bFGF on the inhibitory activity of iota-carrageenan was determined.
  • Figure 14a is a bar chart showing DNA synthesis (as measured by incorporation of tritiated thymidine) in LNCaP cells incubated in the absence (left hand side) or presence (right hand side) of 10ug/ml iota carrageenan.
  • the columns represent (from left to right): no exogenous growth factor; 1ug/ml EGF; 1ug/ml bFGF; and 1ug/ml IGFl.
  • the chart clearly shows that excess bFGF does not reverse the iotacarrageenan-mediated inhibition of DNA synthesis, thus indicating that inhibition of bFGF binding is not primarily responsible for anti-proliferative activity.
  • Figure 15 is a picture of an agarose gel. The contents of the lanes are as follows: 1-phi ⁇ 174 RF DNA/Hae III Digest (0.25ug DNA, reference); 2-DNA from untreated FBHE monolayer cells; 3-DNA from untreated
  • floater detached cells
  • 5-as lane 4 but DNA from "floater” cells
  • TGFalpha transforming growth factor alpha
  • igFI insulin-like growth factor-I
  • PDGF platelet-derived growth factor
  • bFGF basic fibroblast growth factor
  • TGFbeta Transforming growth factor beta
  • ER oestrogen-receptor
  • MCF-7 oestrogen-receptor
  • iota carrageenan was found to inhibit the proliferation of the breast cancer cell line MCF-7. This cell line was therefore selected for further
  • MCF-7 cells and T47D cells were purchased from ECACC.
  • SKBR3 cells were from the American Type Culture Collection (Rockville, Maryland, USA). MCF-7 and SKBR3 cells were maintained in DMEM/10% FCS; T47D cells were maintained in RPMI/10% FCS. All cells received antibiotics (100
  • FCS foetal calf serum
  • DNA synthesis was assessed by measuring the incorporation of a 3-6h pulse of [ 3 H] thymidine into DNA (Dealtry and Balkwill, 1987 cited above). For proliferating cells, 10,000 cells/ml were plated out in medium containing 10% FCS. After a 24h incubation, fresh medium/10% FCS containing carrageenan was added and DNA synthesis was determined 24h later. Determination of the growth factor stimulated DNA synthesis of MCF-7 cells was based on the method of Karey and Sirbasku (1988, Cancer Res. 48, 4083- 4092). 10,000 cells/ml were plated down for 2d in
  • DMEM/10% FCS DMEM/10% FCS.
  • the cells were washed in serum-free DMEM and incubated in serum-free DMEM containing transferrin (10 ug/ml) and BSA (0.2 mg/ml) (defined medium) for 1-2d iotacarrageenan was then added in the defined medium, and 3h later growth factors were added. DNA synthesis was determined after a further 24h.
  • E2 stimulation of MCF-7 cells was determined as follows. 10,000 cells/ml were plated down for 2d in DMEM/10% FCS. Cells were washed with phenol red free DMEM and incubated in phenol red free
  • Figure 16 is a graph of thymidine incorporation against concentration of carrageenan (ug/ml) and shows the results of an initial experiment comparing the effects of a single administration of iota, kappa, or lambda carrageenan on MCF-7 ( Figure 16a) and T47D ( Figure 16b) cells grown in 10% serum.
  • Iota carrageenan is represented by empty circles, kappa carrageenan by filled circles and lambda carrageenan by empty squares. Iota carrageenan partially inhibited the DNA synthesis of MCF-7 and T47D cells when determined 24h after a single administration; kappa and lambda
  • FIG. 18 is a graph of relative thymidine incorporation (%) against concentration of iota carrageenan.
  • Figure 18 shows that DNA synthesis was stimulated by bFGF, IGFl and TGF alpha.
  • the cells did not respond to PDGF (B-chain homodimer).
  • bFGF-stimulated DNA synthesis was inhibited by iota carrageenan to non-stimulated values.
  • TGFalphastimulated DNA synthesis was completely inhibited.
  • IGF-I-stimulated DNA synthesis was either not inhibited or there was only slight inhibition.
  • Iota carrageenan did not significantly inhibit DNA synthesis of the cells not stimulated with exogenous growth factors.
  • iota carrageenan only partially inhibits DNA synthesis of MCF-7 cells grown in serum.
  • Serum contains many mitogenic factors including hormones and growth factors which may be insensitive to iota carrageenan.
  • Tamoxifen has been shown to decrease the responsiveness of MCF-7 cells to mitogenic growth factors and to increase the production of the inhibitory growth factor TGF beta.
  • the present inventors decided to investigate the effects of tamoxifen in combination with iota carrageenan.
  • Figure 20 The results are illustrated in Figure 20 (a-d). Briefly, cells in medium supplemented with 10% serum were treated with various concentrations of iota carrageenan (shown in ug/ml) in the absence (empty circles) or presence (filled circles) of 1uM tamoxifen.
  • Figure 20a shows the results for relative MCF-7 DNA synthesis (as measured by thymidine incorporation)
  • Figure 20b shows the results for relative MCF-7 proliferation (as measured by O.D.)
  • Figures 20c and 20d show relative DNA synthesis for T47D and SKBR3 cells respectively.
  • tamoxifen a combination of tamoxifen and iota carrageenan also significantly enhanced the anti-proliferative activity compared to the activity of either agent alone (Fig. 20b).
  • tamoxifen a combination of tamoxifen and iota carrageenan also significantly enhanced the anti-proliferative activity compared to the activity of either agent alone (Fig. 20b).
  • the ER negative cell line SKBR3 was not inhibited by tamoxifen alone, but iota carrageenan caused more inhibition than in the two ER positive lines, and this inhibition was potentiated by tamoxifen (Fig. 20d).
  • tamoxifen-induced inhibition of DNA synthesis by iota carrageenan is interesting as it occurs at physiologicaly relevant concentrations of the anti-oestrogen.
  • Two ways by which tamoxifen could contribute to the inhibitory activity of iota carrageenan are by reducing the responsiveness of MCF-7 cells to serumderived IGF-like activity (Wosikowski et al., 1993 Int. J. Cancer 53, 290-297) or by inducing TGF beta (Knabbe et al., 1987 Cell 48, 417-428).
  • TGF beta total TGF beta in a thymocyte growth inhibition assay
  • 10 ng/ml TGF beta was required to inhibit DNA synthesis of MCF-7 cells in our experiments and this relatively high concentration is consistent with other workers (Zugmaier et al., 1989 cited previously).
  • 1 ng/ml TGF beta alone did not inhibit DNA synthesis, this concentration did enhance the activity of iota carrageenan. This observation suggests that serum-derived growth factors, sensitive to inhibition by iota carrageenan, may attentuate the anti-proliferative activity of 1 ng/ml TGF beta.
  • carrageenans can inhibit the neovascularisation which is essential for tumours to progress beyond a certain limited size, thereby inhibiting tumour growth indirectly.
  • carrageenans can inhibit tumour growth directly by interfering with growth factor binding and subsequent growth "signals".
  • the mechanism(s) have not been fully elucidated, but it seems possible that carrageenans block the autocrine and paracrine growth factor stimulation of several different tumour cells.
  • This inhibition can be potentiated by other growth antagonists, such as the anti-oestrogen tamoxifen, which block other growth signal pathways insensitive to the action of carrageenans.
  • Activated partial thromboplastin time was determined using
  • Figure 21 is a graph of clotting time (in seconds) against concentration of polysulphated polysaccharide (in ug/ml). Data points are as follows: heparin (filled circles), lambda carrageenan (boxes), kappa carrageenan (crosses) and iota carrageenan (5-pointed stars).
  • carrageenan derivatives A range of lower molecular weight derivatives of iota carrageenan were prepared using the novel method devised by the instant inventors, described below.
  • Carrageenans e.g. iota carrageenan, with a molecular weight in the range 200 - 500, kD
  • This solution was dialysed against distilled water to remove low molecular weight impurities (with a molecular weight less than 10kD).
  • the product is a white powder and is the low molecular weight form of free acidic carrageenans (mw 0-125kD).
  • Carrageenans freeze dried from a solution with a pH below 4.0 are unstable and become degraded, giving a range of lower molecular weight products.
  • the pH of the carrageenan solution is adjusted to about pH7.0 with sodium hydroxide. Freeze drying as above gives the sodium salt.
  • This method is preferable to, for example, chemical or enzymatic hydrolysis as it is particularly "gentle", leaving the anhydrogalactose residues and sulphate groups unaltered. Also, chemical hydrolysis is inefficient due to the high viscosity of carrageenan solutions.
  • the various products can be fractionated according to their charge and/or molecular weight.
  • fractions can be characterised by various conventional techniques. For example, molecular weight and molecular weight
  • the galactose and anhydrogalactose content can be assayed by gas chromatography; and the sulphate content can be measured by a conductometric or photometric method.
  • Figure 22a shows the amount radiolabelled bFGF bound to low affinity receptor sites in the presence of 100 ug/ml (solid bar) or 10ug/ml (hatched bar) of each fraction.
  • Figure 22b shows the results for bFGF binding to high affinity receptors (same symbols as for Figure 22a).
  • Figure 23 shows the amount of DNA synthesis as measured by cpm of tritiated thymidine incorporation in FHBE cells incubated in the presence of 100ug/ml (solid bars) or 10ug/ml (hatched bars) of each fraction.
  • fractions showed inhibitory activity when present at 100ug/ml, especially fraction 67, although no fraction was as active as iota carrageenan itself.
  • Fraction 67 was prepared by ion exchange chromatography on a strong anionic column, using a 0-4M NaCl gradient. The fraction has a molecular weight range of 3,200-105,000

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Abstract

L'invention se rapporte à une substance composée d'un carraghénane ou d'un de ses dérivés, utilisée comme inhibiteur ou suppresseur de facteur de croissance. Les substances décrites comme pouvant être utilisées comme inhibiteurs de facteur de croissance conviennent au traitement de tumeurs et autres états auxquels sont associés les facteurs de croissance.
PCT/GB1993/001848 1992-09-01 1993-09-01 Utilisation de carraghenanes comme antagonistes de facteur de croissance Ceased WO1994005267A2 (fr)

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AU49726/93A AU4972693A (en) 1992-09-01 1993-09-01 Use of carrageenans as growth factor antagonists

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GB929218510A GB9218510D0 (en) 1992-09-01 1992-09-01 Improvements in or relating to hormone antagonists
GB9218510.7 1992-09-01

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WO1994005267A2 true WO1994005267A2 (fr) 1994-03-17
WO1994005267A3 WO1994005267A3 (fr) 1994-04-28

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6294195B1 (en) 1991-12-24 2001-09-25 Purdue Pharma L.P. Orally administrable opioid formulations having extended duration of effect
FR2808687A1 (fr) * 2000-04-27 2001-11-16 Goemar Lab Sa Medicament contenant des substances polysaccharidiques pour l'activation de l'apoptose

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US4783446A (en) * 1985-11-22 1988-11-08 Neushul Mariculture Incorporated Method for the treatment of AIDS virus and other retroviruses

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6294195B1 (en) 1991-12-24 2001-09-25 Purdue Pharma L.P. Orally administrable opioid formulations having extended duration of effect
FR2808687A1 (fr) * 2000-04-27 2001-11-16 Goemar Lab Sa Medicament contenant des substances polysaccharidiques pour l'activation de l'apoptose
WO2001080807A3 (fr) * 2000-04-27 2002-04-18 Goemar Lab Sa Medicament contenant des substances polysaccharidiques pour l'activation de l'apoptose

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AU4972693A (en) 1994-03-29
WO1994005267A3 (fr) 1994-04-28
GB9218510D0 (en) 1992-10-14

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